Background: Osteoporosis is a common orthopedic disease with high prevalence in patients older than 50 years. Osteoporosis is often detected only after the fracture and is hard to treat. Therefore, it is of great significance to explore the molecular mechanism of the occurrence of osteoporosis.Methods: The expression of Heme oxygenase-1 (HO-1) in people with different bone mineral density (BMD) was analyzed based on public databases. GenHacncer and JASPAR databases were adopted to search and verify the upstream transcription factor of HO-1. qRT-PCR, western blot and tartrate-resistant acid phosphatase assays were performed to explore the impact of HO-1 and Kruppel-like factor 7 (KLF7) on osteoclast differentiation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding relationship between KLF7 and HO-1. Finally, Hemin, the agonist of HO-1, was applied in rescue assays, thereby verifying the mechanism of KLF7 modulating osteoclast differentiation by HO-1.Results: Bioinformatics analysis revealed that HO-1 was highly-expressed while KLF7 lowly-expressed in people with high BMD. Besides, a potential binding site of KLF7 was found on the promoter region of HO-1. ChIP assay further manifested the targeting relationship between HO-1 and KLF7. Western blot and TRAP staining unveiled that osteoclast differentiation was suppressed by HO-1, while facilitated by KLF7. Rescue experiments indicated that over-expressed HO-1 could reverse of the promoting effect of KLF7 on osteoclast differentiation.Conclusion: The study confirmed that osteoclast differentiation was promoted by KLF7 constraining HO-1, thereby facilitating osteoporosis. The cognation of the pathogenesis of osteoporosis was further enriched. New treatment could be developed on this basis. Osteoporosis is a common orthopedic disease, which is characterized by low bone mineral density (BMD), the deterioration of bone microarchitecture and the increase of fracture occurrence . AccordiHeme oxygenase-1 (HO-1) is known as a negative regulator of osteoclast differentiation and is involved in the process of bone renewal and repair. Generally, Hrf2/HO-1 pathway is considered to be a key pathway that influences the occurrence of osteoporosis . PreviouKruppel-like factor 7 (KLF7) is a transcription factor (TF) that accelerates the regeneration in the central nervous system axon . It was Based on previous studies and bioinformatics results, it could be postulated that HO-1 and KLF7 could regulate the osteoclast differentiation. Molecular and cell experiments were performed to prove that KLF7 accelerates osteoclast differentiation by suppressing HO-1, thereby the development of osteoporosis was affected. The result of this study disclosed the molecular mechanism of KLF7/HO-1 in osteoporosis, which provides theoretical basis for clinical treatment and drug development of osteoporosis.2 at a constant temperature of 37°C. Recombinant mouse macrophage colony stimulating factor  and recombinant mouse receptor activator of nuclear factor kappa-B ligand  proteins were used to activate RAW264.7 to differentiate into osteoclasts. In short, after the cells in the medium have grown to an appropriate level, the medium was replaced with the medium containing 30 ng/ml M-CSF and 50 ng/ml RANKL. Cells were then used for subsequent assays after being incubated for an appropriate time. Hemin (HO-1 activator) was added into the medium at a final concentration of 10 μM. After 8 h of culture, M-SCF and RANKL were used to activate macrophage differentiation in accordance with the above processes.Murine mononuclear macrophage leukemia cell line RAW264.7 (BNCC337875) was purchased from BeNa Culture Collection . The cells were cultured in 90% Dulbecco’s modified eagle medium (DMEM) + 10% fetal bovine serum (FBS)and then placed in an incubator with 5% COhttps://www.ncbi.nlm.nih.gov/gds/), including data of 40 females with low BMD and 40 females with high BMD. The subjects were classified into high and low BMD groups according to their BMDs. Then limma package was used for differential expression analysis and data standardization. Gene expression was visualized using Graphpad Prism. T-test was utilized to analyze the significance of the differences between gene expression in the two groups.Osteoporosis-related gene expression data of blood leukocytes were downloaded from GSE56815 dataset (https://www.genecards.org/Guide/GeneCard#enhancers). These predicted TFs were intersected with differentially expressed TFs in osteoporosis. The expression of acquired TFs and HO-1 were subjected to Pearson correlation analysis. Thereafter, the gene significantly correlated with HO-1 was adopted as the object of the study.TFs involving in the upstream regulation of HO-1 were predicted using GenHancer database (http://jaspar.genereg.net/) was employed to predict the binding relationship between the predicted target TF and HO-1. Firstly, the 5’ end sequence extending from the transcription start site (TSS) region at 2,000 bp in the upstream of HO-1 gene was downloaded from National Center of Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Thereafter, this sequence was compared with the TF in Jaspar database. The result was determined as the possible binding site on the TF.Jaspar database . 5 ul of Lipofectamine3000  was used to transfect 50 nM si-HO-1 and oe-KLF7/si-KLF7 into cells. Successfully transfected cells were collected after 72 h.4/well), followed by a fixation with 10% formaldehyde solution. TRAP staining kit  was applied to stain the inoculated cells, after which the stained cells were observed under a microscope. A single colony with more than 3 cells whose nucleus was stained (TRAP-positive) was counted as an osteoclast.The treated cells were collected and inoculated into 24-well plate  for qPCR detection. GAPDH was used as internal reference of the obtained data. The relative expressions were calculated using 2−ΔΔCT method for subsequent analyses. TRIzol kit  was used for the extraction of total RNA from the cells based on the instructions of the manufacturer. The concentrations of extracted RNA were assessed utilizing NanoDrop™ One  and then diluted to an equal level. Afterwards, QuantiTect Reverse Transcription Kit  was employed to reversely transcribe RNA into cDNA which were then used for qPCR detection proceeding with SYBR™ Green PCR Master Mix . In brief, 1 ng cDNA, appropriate dose of primers, ddHAfter the cells were treated in accordance with experimental design, they were collected for the extraction of total proteins using radioimmunoprecipitation assay (RIPA) lysis buffer. The content of total protein was measured using bicinchoninic acid (BCA) kit. After the proteins were extracted, 30 μg total proteins were placed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for isolation. Thereafter, the proteins were transferred onto a polyvinylidene fluoride (PVDF) which was then blocked with 5% nonfat-dried milk. Afterwards, the membrane was sequentially incubated with primary anti-HO-1 , anti-GAPDH , anti-NFAT2 , anti-TRAP , anti-CTSK, anti-KLF7  and corresponding secondary antibody . The concentration of the proteins was gauged utilizing electrochemiluminescence (ECL) luminescence reagent. Each assay was repeated three times.First of all, RAW264.7 cells were transfected using pCMV-Myc-KLF7. After 24 h of transfection, anti-Myc antibody  and corresponding Simple ChIP enzymatic chromatin IP kit  were subjected to ChIP assay following the instructions of the manufacturer. In the end, depurated DNA was detected using qPCR. The primers used for detections were as follows .T-test was utilized for differential evaluations among groups. p < 0.05 denoted a statistical significance.All data in this study were denoted as MEAN ± standard deviation (SD). GraphPad Prism and R software were adopted for analysis and drawing. Analysis of variance and Student’s Firstly, patients’ gene expression data in GSE56815 dataset were downloaded. It was found that the expression of HO-1 was remarkably up-regulated in females with high BMD in comparison with the ones with low BMD. . In combTo dissect the factor that affects HO-1 expression and explore potential therapeutic target of osteoporosis, we used bioinformatics approaches to investigate the regulator in the upstream of HO-1. GenHancer was first adopted to predict the TFs that might affect expression of HO-1. Then the expression data of GSE56815 were downloaded, and 358 differentially expressed genes were screen out . DiffereUnderlying the results of bioinformatics analysis, we speculated that KLF7 could also influence osteoclast differentiation. In support to our speculation, we first constructed si-KLF7 and oe-KLF7 plasmids to processed the cells. The outcome indicated that the knockdown of KLF7 expression led to a marked decrease in mRNA and protein expressions of KLF7, whereas it had an opposite impact on those of HO-1 . BesidesAfter the possible effects of KLF7 on osteoclast differentiation were clear, we further validated the binding relationship between TF KLF7 and HO-1. First of all, we separately used hemin and oe-KLF7 to process the osteoclasts. Hemin enhanced HO-1 expression, whereas oe-KLF7 could reverse this effect. . Based oThe clinical treatment of osteoporosis mainly includes calcium and vitamin-based nutrients, estrogen receptor modulators, disphosphonates, and anti-RANKL drugs, etc. . AlthougHO-1 is a key gene that is usually found to affect the damage repair of the bones. Many studies have investigated the impact of HO-1 on osteoporosis. Studies proved that osteoclast differentiation is among various factors that affect osteoporosis . For exaThe regulation of TF is the commonest regulatory form of gene expression, and the related regulatory relationship of transcription factor is regarded as a key factor that affects the occurrence of osteoporosis. For instance, TFs like AP‐1 and Mitf can impact the activity of NF-kB pathway by modulating NFATc1 and then induce macrophage to differentiate into osteocyte . MoreoveThe results of this study confirmed that the TF KLF7 could induce osteoclast differentiation by inhibiting HO-1, thereby promoting the development of osteoporosis. Our study provides a novel possible target for the development of osteoporosis drugs, which is of great clinical value. Although cell experiments have proved the role of KLF7 in osteoclast differentiation, but it has not been verified at animal level, which is a shortcoming of this study. Therefore, we plan to carry out further animal experiments to further explore the therapeutic value of KLF7 in osteoporosis.


MECP2 gene are the primary cause of Rett syndrome and, to a lesser extent, of a range of other major neurodevelopmental disorders. Here, we aim to study the interaction of MeCP2 with early-life stress in variables known to be affected by this environmental manipulation, namely anxiety-like behavior and activity of the underlying neural circuits.Early-life stress can leave persistent epigenetic marks that may modulate vulnerability to psychiatric conditions later in life, including anxiety, depression and stress-related disorders. These are complex disorders with both environmental and genetic influences contributing to their etiology. Methyl-CpG Binding Protein 2 (MeCP2) has been attributed a key role in the control of neuronal activity-dependent gene expression and is a master regulator of experience-dependent epigenetic programming. Moreover, mutations in the Mecp2 heterozygous and wild-type female mice we investigated the effects of the interaction of Mecp2 haplodeficiency with maternal separation later in life, by assessing anxiety-related behaviors and measuring concomitant c-FOS expression in stress- and anxiety-related brain regions of adolescent females. Moreover, arginine vasopressin and corticotropin-releasing hormone neurons of the paraventricular hypothalamic nucleus were analyzed for neuronal activation.Using Mecp2 heterozygous females that per se show decreased anxiety-like behavior and concomitant decreased paraventricular nuclei activation.In wild-type mice, maternal separation caused a reduction in anxiety-like behavior and in the activation of the hypothalamic paraventricular nucleus, specifically in corticotropin-releasing hormone-positive cells, after the elevated plus maze. This effect of maternal separation was not observed in Our data supports that MeCP2 is an essential component of HPA axis reprogramming and underlies the differential response to anxiogenic situations later in life. Early-life stress (ELS) constitutes a major risk factor for the development of psychiatric disorders, including anxiety disorders. These disorders are the most frequent mental disorders and impose an enormous societal and economic burden , 2. HereMeCP2 is a multifaceted protein with roles in transcriptional regulation, epigenetic programs, alternative splicing, microRNA processing and chromatin remodeling, thereby facilitating several biological processes . In partThe activity of the hypothalamic-pituitary-adrenal (HPA) axis is characterized by a prominent critical period of plasticity . AdverseMECP2 gene are the major cause of Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects females [MECP2 mutations are also responsible by other major neurodevelopmental disorders such as X-linked mental retardation, autism, Angelman syndrome or schizophrenia [MECP2 duplication syndrome (MDS), that affects mainly males, show intellectual disability and autism [Loss-of-function mutations in the  females . In its  females . Males u females . To a leophrenia . By contd autism . The invImportantly, RTT patients show a blunted decrease of around 50% of cortisol concentration from morning to evening, as compared with typical values of 90% decrease in healthy individuals . InteresCrh gene is a direct target of MeCP2 [Crh [Several different genetic mouse models have been generated that mimic the major human behavioral, neuroanatomical, and biochemical aspects of the different disorders (reviewed in ), allowiof MeCP2 , 23 and CP2 [Crh , 22, 24.MECP2-related disorders. On the other hand, MECP2 gene can itself constitute a genetic risk factor for the development of anxiety and stress-related disorders, though future studies are needed to investigate this later possibility.In this scenario, it is plausible that decreased functionality of MeCP2 early in life, as that seen in RTT patients, will impact the epigenetic programming of the HPA axis thereby affecting their vulnerability to develop anxiety and stress-related disorders later in life, when encountering a stressor. In fact, anxiety-like behavior is a prominent component of RTT’s behavioral phenotype . WhereasMecp2-deficient mice have not been experimentally tested. In this study, we used Mecp2 heterozygous (Mecp2.het) and wild-type (WT) female mice that were, or were not, submitted to maternal separation (MS), a classical rodent paradigm to study ELS [To our knowledge, the effects of ELS in anxious behavior in tudy ELS , 30. Inttudy ELS , 31. Thetudy ELS . FinallyMecp2.het females and their wild-type littermates from our colony. To establish the colony, we purchased Mecp2.het females from Jackson Laboratory -Mecp2tm1.1Bird/J) [For this study, we used the 1Bird/J)  and mainAll the procedures were carried out in strict accordance with the EU directive 2010/63/EU. The protocols were approved by the Ethics in Animal Experimentation Committee of the University of Valencia.n = 13; Mecp2.het-MS, n = 18) were separated from the dam as a group, and kept in a new cage filled with sawdust and warming red light for 3 h per day, after which pups were returned to their home cage. Control animals were included  that were maintained undisturbed with their dams in the home cage until weaning. All these animals were subjected to the battery of behavioral tests.To induce ELS we used the MS protocol. From PND3 to PND21 pups  were allowed to explore a squared arena (50 cm × 50 cm × 50 cm) in a 20-min session, under red light illumination. The total distance travelled was used a measure of activity and the distance travelled and time spent in a predefined center area (25 cm × 25 cm) versus the rest of the arena was used to assess anxiety-like behavior. Movement of mice was tracked using an automated tracking system .Animals  were put in an inescapable transparent jar filled with water and allowed to swim for a 5-min period. The time animals spent immobile was measured. Mice were dried out in paper towel before they were returned to their home cage.To assess depressive-like state mice were tested in the FST. Mice . The first EPM session was the first test of the behavioral battery and was performed under red light conditions. The second EPM session was the last test of the battery and was performed under bright light, aversive conditions, and 1 h after the test animals were sacrificed. The maze consisted of two opposing closed arms  and two opposing open arms  extending from a common central region (6 × 6 cm) to form a ‘plus’ shape. The arms were elevated 40 cm above the floor. In a single 5-min trial, animals were placed in the center of the maze and the time spent, distance travelled and number of entries in the open and closed arms were recorded. Movement of mice was tracked using an automated tracking system .Mice were tested in the EPM in two sessions  were deeply anaesthetized 1 h after the last EPM session (bright conditions), using a mixture of ketamine (75 mg/kg) and medetomidine (1 mg/kg) and transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer . Brains were carefully removed from the skull, post-fixed in the same fixative for 4 h and placed into a 30% sucrose solution in 0.1 M phosphate buffered saline  until they sank. The brains were then frozen and cut in six series of 40-μm-thick coronal sections with a freezing microtome. Free-floating sections were frozen in 30% sucrose in PB/0.02% sodium azide (0.1 M pH 7.4) for their posterior processing.A random subset of the animals used in the behavioral battery was selected for anatomical studies. Animals   for 30 min at room temperature (RT). Next, sections were incubated in blocking solution containing 3% normal goat serum (NGS) in TBS-triton X100 (Tx) 0.3% for 1 h at RT. Then, sections were incubated in rabbit anti-c-FOS  in TBS-Tx with 3% NGS 24 h at 4 °C, followed by an incubation with biotinylated goat anti-rabbit IgG  in TBS-Tx with 2% NGS for 2 h at RT. Afterwards, sections were incubated in ABC Elite  in TBS-Tx for 90 min at RT. Finally, the resulting peroxidase labelling was revealed with 0.025% 3-3′-diaminobenzidine  in PB  and 0.01% H2O2 for 25 min. Sections were mounted onto gelatinized slides, dehydrated in alcohols, cleared with xylene and cover slipped with Entellan .One out of six brain slices parallel series of each subject was processed for the immunohistochemical detection of c-FOS. To do so, endogenous peroxidase was inactivated with 1% HWe obtained double immunofluorescent labelling for c-FOS/AVP and c-FOS/CRH in two out of six brain slices parallel series of each subject. Brain slices were first incubated with 1% sodium borohydride in 0.05 M TBS for 30 min at RT to block the endogenous fluorescence of the tissue. Next, sections were incubated in a blocking solution containing 3% normal donkey serum (NDS) in TBS-Tx 0.3% for 1 h at RT. Afterwards, each series was incubated for 48 h at 4 °C with a mixture of primary antibodies containing rabbit anti-AVP  or rabbit anti-CRH  and goat anti-c-FOS  in TBS-Tx 0.3%/ NDS 2%. Next, brain slices were incubated for 90 min at RT with appropriate secondary antibodies Rhodamine Red-X Donkey anti-Rabbit  and Alexa Fluor® 488 Donkey Anti-Goat  in TBS-Tx0.3% with 2% NDS. Finally, sections were mounted onto gelatinized slides and cover-slipped with fluorescence mounting medium .After c-FOS immunohistochemistry, we quantified c-FOS-positive nuclei in representative levels  were obtained using Leica TCS SPE confocal microscope. Then, an experimenter blind to the condition of the animals counted the number of c-FOS-positive nuclei using the ImageJ Cell Counter analysis tool and co-expression of these nuclei with either AVP or CRH was verified visually and the number of double immunoreactive cells registered.Mecp2.het) as between-subject sources of variance, followed by post hoc Bonferroni correction for multiple tests. Significance was set at p < 0.05. Data were analyzed using the software IBM SPSS Statistics 22.0.Behavioral and histological data were analyzed with two-way ANOVA with treatment (naïve or MS) and genotype , naive and MS groups, were assessed for anxiety-like and depressive-like states to study the effects of Mecp2 and ELS interaction of in the vulnerability of animals to adversity later in life. Thus, experimental subjects were submitted to tests starting with the EPM, OF, FST, and finishing with a second EPM performed in bright light conditions, 1 h after which animals were sacrificed to assess c-FOS activation patterns.WT and F = 8.633, p = 0.0049) in which Mecp2.het females spent more time in the open arms than WT animals  in a single 5-min session. No differences were observed among groups in the total distance travelled in the maze Fig. A. HoweveF, 53 = 8.F, 53 = 8.F = 8.461, p = 0.0053; Fig. Mecp2.het females travelling a smaller total distance than their WT controls. Regarding time spent in the center of the apparatus in relation to the remaining area, we did not find a statistically significant effect of any factor  = 27.09 p < 0.0001; Fig. Mecp2.het females have lower immobility times than WT animals, indicative of an antidepressant-like phenotype. In addition, we also observed a trend towards significance in group x genotype interaction effect  = 3.023 p = 0.0881; Fig. Mecp2.het females have lower immobility times than naïve WT animals  = 4.757 p < 0.0001). In the MS groups, no statistically significant differences in immobility times were detected between WT and Mecp2.het females  = 2.625 p = 0.0684; Fig. Mecp2.het females per se occludes this phenotype. Although we do not observe an effect of MS in Mecp2.het females, we cannot exclude that it can be masked by a floor effect given the already very low immobility time in the Mecp2-naive group.The performance of WT and effect  = 9.095; p = 0.0039). Additionally, MS significantly increased the percentage of time spent in the open arms as compared with naïve animals  = 9.304; p = 0.0036; Fig. Mecp2.het females exposed to MS show lower levels of anxiety in face of an anxiogenic situation.Finally, all animals were tested in a more anxiogenic version of the EPM, under bright light conditions. Here, no differences were observed in the total distance travelled among groups  = 5.801 p = 0.028). Post hoc tests showed that MS reduced the number of c-FOS-positive nuclei in WT animals  = 24.453 p < 0.001). In addition, Mecp2 deficiency resulted in a strong reduction of c-FOS-positive nuclei, as that seen in the Mecp2.het-naive females, when compared to WT-naive animals  = 14.520 p = 0.002) and MS did not significantly add up to the effect (p > 0.5).Mice were sacrificed 1 h after exposure to the bright EPM to allow for subsequent quantification of c-FOS-positive nuclei in brain regions (i) known to be activated after EPM exposure and (ii) related to stress and anxiety processing . In the F = 5.382 p = 0.032) and genotype  = 6.990 p = 0.017). Overall, MS resulted in a reduction of c-FOS-positive nuclei, particularly noticed in WT animals, and Mecp2.het females had a lower number of c-FOS-positive nuclei than WT animals  = 4.920 p = 0.044; Fig. F = 13.461 p = 0.003). Further, Mecp2 deficiency per se resulted in a strong reduction of c-FOS/CRH double-labeled neurons  = 8.740 p = 0.010; Mecp2.het-naive vs Mecp2.het-MS, p > 0.5).Since the most significant effects of both ons Fig. A–D. Datap > 0.5). Previous studies suggested and increase in Avp expression levels after MS [p = 0.055). On the other hand, the density of AVP cells in the Pa was not affected by Mecp2 genotype (data not shown), in agreement with our previous study [By contrast, no statistically significant effects of either factor were found for the density of c-FOS/AVP double-labeled neurons  and behavior later in life. In the current study, we show that animals that were maternally separated (MS groups) display a reduction in the anxiety levels when tested in the bright EPM.Mecp2 haplodeficiency per se resulted in low levels of anxiety, mimicking the MS effects. In its first description, Mecp2.het females were reported as being asymptomatic at young ages, taking up to 6 months of age for 50% of females to develop overt symptoms [Mecp2.het pups have a delay in the development and acquisition of neurological reflexes [Mecp2 haplodeficiency at such young ages could affect the development of critical periods, and have an impact later in life. Here, we provide evidence that adolescent Mecp2.het females show indeed an altered sensitivity to early-life stress. In particular, at 6 weeks of age, Mecp2.het females present with less anxiety and less depressive-like behaviors, as compared to WT animals. Our data agrees with a previous study showing decreased anxiety in the EPM of 5 weeks old Mecp2.het females of mixed background [Mecp2.het females of the strain used in the present study. However, other studies using Mecp2-deficient models have reported conflicting results when assessing anxiety-like behavior.Previous studies demonstrated that MeCP2 is one of the factors mediating the effects of ELS in cognitive and emotional behaviors in mice . Importasymptoms . Later treflexes , suggestckground . More reckground  also repMecp2.het females might contribute the genetic background of the dam by affecting maternal behavior, which in turn will affect pup’s response to a stressor [Mecp2.het dams produce smaller litters and often neglect and cannibalize their offspring [Mecp2 genotype and/or strain, should be considered to clarify the effects here observed. Cross-fostering with CD-1 strain, which are good breeders, has actually proven beneficial in the evaluation of other RTT-like phenotypes [Mecp2+/− females. These results warrant future studies disentangling the type and time windows of environmental interventions that could be beneficial to ameliorate RTT phenotypes.To the reduction of the anxious phenotype of stressor , 39. In ffspring , in addiffspring . In factffspring . Altogetenotypes . Moreoveenotypes  showed tMecp2.het questions that this phenotype is solely due to anxiety alterations. Strikingly, Flores Gutiérrez et al. [Mecp2.het females with trimmed whiskers did not show the anxiolytic phenotype in the EPM, suggesting that sensory hypersensitivity could be an important contributor of lowered anxiety. Nonetheless, our results showing decreased activation of the CRH neurons of the paraventricular nucleus indeed support a reduced anxiogenic response (see below). Taken together, these results reflect the complexity of assessing the causes of aberrant behaviors in both mouse models and RTT patients, given the sensory and motor impairments that characterize the disease. In the case of patients, this is further complicated by communication impairments. At the same time, our data supports the use of young Mecp2.het, largely pre-symptomatic females, as a valid model to study non-motor phenotype alterations.A recent study in 6 months old, symptomatic, z et al.  found thMecp2.het females show low numbers of c-FOS neurons in the same regions, and MS has no additional effect. Our results show that MS precludes the activation of Pa and PV neurons, normally activated upon EPM exposure, and indicate that this may be a MeCP2-dependent process, as deficiency of Mecp2 per se also precludes the activation of such neurons.EPM is an anxiogenic stressor and exposure to the maze induces neuronal activation in several brain areas associated with stress and anxiety , 45. In Crh, as rats subjected to MS showed reduced occupancy of several transcription factors, including MeCP2, in the promoter region of Crh gene accounting for its upregulation [Mecp2 in hypothalamic neurons decreases Crh mRNA in the Pa [Mecp2308 mouse model, expressing a mutated form of MeCP2, presents with heightened anxiety and elevated levels of Crh expression in the hypothalamus [Crh gene, which associates with elevated levels of Crh expression [Crh gene expression, as MeCP2 knockdown leads to increased expression of Crh [Crh and displayed heightened anxiety-like behavior when subjected to the EPM and to the light-dark box [Mecp2 mutation type could also account for the apparently contradictory effects of MeCP2 on anxiety.The Pa is a stress-responsive brain region and a key center of the HPA axis, containing CRH neurons that control adrenocorticotrophic hormone (ACTH) release from the hypophysis, which in turn activates the adrenal release of corticosterone to the bloodstream. ELS induces activity-regulated gene expression and alters the distribution of epigenetic marks, which MeCP2 reads, thereby modulating gene expression of a specific set of genes later in life , 7. One gulation . Our resn the Pa . Insteadthalamus , 20, 24.pression . Indeed,n of Crh . On the dark box . The infdark box . NeverthCrh expression in Mecp2-deficient models suggest that feedback loops ensuring the correct function of HPA axis are compromised in Mecp2 mouse models. Consistent with this, modulation of the corticosteroid levels has proven beneficial in these models with a positive impact in locomotor/exploratory behavior, motor symptoms and lifespan [Mecp2.het females. In line with our results, Mecp2+/− females in a mixed background are less anxious and have an abnormal corticosterone response to stress, either with no differences or with decreased levels of corticosterone, when compared to control animals [308 females have lower levels of corticosterone than wild-type females.Our data showing reduced neuronal activation of CRH neurons, together with other studies showing a decrease  or increlifespan , 48. The animals . Moreove animals , using aMecp2 deficiency in the activation of AVP neurons. Previous data showed that MS induced hypomethylation at the Avp enhancer region, and a low presence of bound MeCP2 in the same region, thereby increasing Avp expression specifically in the Pa [Mecp2308 null), but not mice with MeCP2 haplodeficiency (Mecp2308 het), show an increased expression of Avp in the hypothalamus [Mecp2 deficiency had no effect in the density of AVP neurons in the Pa, in line with our previous results [Mecp2 in hypothalamic neurons [Our data did not reveal a statistically significant effect of either MS or n the Pa . Also, mthalamus . In our  results  and with neurons .Mecp2.het females might reflect, as in the case of Pa, an inability to appropriately respond to an anxiogenic situation. Finally, although we did not find a statistically significant effect in the BSTLD and LSV, these structures display a trend towards hypoactivation in both Mecp2.het and WT-MS females, similar to what was found in Pa and PV. This trend was not seen in the in the DG, suggesting that differences observed in c-FOS induction after EPM are specific of the emotional circuits.Regarding the PV, this thalamic nucleus is a key hub of the circuits regulating emotional behaviors, and displays important connections with the hypothalamus, BST, and amygdala . Indeed,MECP2 constitutes a shared genetic risk factor to multiple neurodevelopmental disorders; primarily to RTT [per se a risk factor for the development of psychopathology [Mecp2 mouse models [Mecp2308/Y mice show susceptibility to develop PTSD-like symptomatology after a traumatic event, reinforcing the role of MeCP2 in psychopathology.y to RTT  and, to y to RTT , schizopy to RTT , or intey to RTT  and to My to RTT . Importaathology , MDS [28e models , 22. In e models  showed tMecp2.het females. Future studies using models that allow a spatiotemporal control of MeCP2 expression should be used in order to prove its involvement and explore the underlying molecular mechanisms. It is unquestionable that changes in MeCP2 expression affect anxiety-like behaviors. However, further investigation is needed to fully elucidate the anxiety profile in RTT and MECP2-associated diseases. In addition, it is possible that there is currently an underreported association between MECP2, anxiety  and stress-related disorders (posttraumatic stress disorder), and future studies in the field should screen for MECP2 variants and also methylation levels (mCA and mCpG) of specific MeCP2 target genes , which early in development are sensitive to the environment and modulate the response to stressors later in life.Data presented in this study suggest that deficiency of MeCP2 early in development, through the regulation of the critical period of HPA axis, may underlie the alterations in anxiety behavior observed in adolescent Mecp2-dependent mechanism is involved in controlling neuronal activity in stress-related circuits that are also responsive to early-life adversity. Moreover, data confirmed CRH pathway as a potential entry point for the treatment of anxiety in RTT and related conditions.In conclusion, this study provides evidence that a


Long intergenic non-protein coding RNA 882 (LINC00882) are abnormally expressed in several tumors. Our research aimed to uncover the functions and the potential mechanisms of LINC00882 in hepatocellular carcinoma (HCC) progression.In vivo tumor xenograft models were constructed to assess tumorigenicity. RT-PCR, Western blot and Luciferase reporter assays were conducted to examine the regulatory relationships among LINC00882, miR-214-3p and ATF2.RT-qPCR was applied to identify LINC00882 and miR-214-3p levels in HCC specimens and cells. Luciferase reporter was applied for the exploration of whether activating transcription factor 2 (ATF2) could bind to the promoter region of LINC00882. Cell proliferation, invasion, and migration were evaluated. LINC00882 was markedly upregulated in HCC cells and clinical specimens. Additionally, ATF2 could bind directly to the LINC00882 promoter region and activate its transcription. Loss-of-function studies further demonstrated that LINC00882 knockdown inhibited proliferation, invasion, and migration of HCC cells. Mechanistically, LINC00882 adsorbed miR-214-3p, thus promoting the expressions of CENPM. Rescue assays demonstrated that functions of LINC00882 deficiency in HCC cells were reversed through suppressing miR-214-3p.Our group identified a novel regulatory axis of ATF2/LINC00882/miR-214-3p/CENPM, which may provide potential therapeutic targets for HCC. Hepatocellular carcinoma (HCC) is one of the most common malignant cancers . It has Long noncoding RNAs (lncRNAs) are RNAs with > 200 nucleotides in length without protein coding capacities . They arLong intergenic non-protein coding RNA 882 (LINC00882), located on 3q13.12, was a new cancer-related lncRNA . SeveralA total of 6 pairs of tumor specimens and the matched non-tumor specimens were collected from National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital. Chemotherapy or radiotherapy were not used for all patients before the operation. All patients gave informed consent and signed a written consent form. This research was ratified by the Ethics Review Committee of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital.2 at 37°C.The normal liver cells (HepaRG) and HCC cell lines  were purchased from ATCC. HCC cells were cultured in a RPMI 1640 medium containing 10% fetal bovine serum (FBS) and HepaRG cells in keratinocyte-SFM , together with 100 U/mL penicillin and 100 μg/mL streptomycin with 5% COShort-hairpin RNA oligos directed against LINC00882 (sh-LINC00882-1 and sh-LINC00882-2) were synthesized. Then, oligos were ligated into the shRNA vectors . To increase the expressions of ATF2 in HCC cells, the expressing plasmids for ATF2 were PCR-amplified and subcloned into the pcDNA3.1 vector, respectively . An empty pcDNA 3.1 served as a control. Ruibio Biology provided miRNA-214-3p mimics and miRNA-214-3p inhibitors. ATF2 small interfering RNA (si-ATF2-1 and si-ATF2-2) and the corresponding control RNA (si-NC) were purchased from Biomics Bio  Co., Ltd. For cellular transfection, Lipofectamine 3000  was applied based on the operating instructions.® Premix Ex Taq ™ and StepOne Plus Real-time PCR System were applied to perform RT-PCR reactions. The primers were shown in Total RNAs in specimens and several cells were extracted by TRIzol reagent . Then, total RNAs were synthesized to cDNA by the use of PrimeScript RT reagent Kit  based on the operating instructions. SYBRNuclear and cytoplasmic separation was carried out using the PARIS Kit  based on the operating instructions.2 for 24, 48, 72 and 96 h, respectively. CCK-8 was used to incubate the above cells for 4 h. Using a microplate reader, the absorbance was observed at 450 nm.Transfected Huh7 and HepG2 cells (4000 cells/well) were incubated at 37°C in an atmosphere with 5% CO3 per well) were seeded in six-well plates. Subsequently, 4% paraformaldehyde  was applied to fix formed colonies for 30 min. 0.1% Crystal violet dye  was used to stain the cells for 15 min. Finally, our group counted and photographed the colonies.Cells (at a density of 3 × 103) were placed in 48-well plates. Under the condition of 5% CO2 and 37°C, 50 μM of EdU diluent was used to cultivate cells for 2 hours. Then, PBS was used rinse the collect cells which was fixated by the use of paraformaldehyde. Next, Apollo 567 working solution was applied to process cells. For the counterstain of the nuclei, DAPI was added for 8 minutes. Finally, fluorescent microscopy was used to observe the collected cells.EDU experiments were conducted for exploring cellular capabilities. Cells  was applied for the observation of scratch distances.Cellular scratch experiments were applied for the determination of the effects of LINC00882 on invasive abilities. Briefly, 1×10The transwell assays were conducted to examine cellular invasion. Briefly, Huh7 and HepG2 cells were seeded on the top transwell chamber  to the bottom chamber, 500 μL of medium was added. 90% ethanol was applied to fix the cells after 24 h. Besides, 0.5% crystal violet prior was applied to stain the collected cells. An inverted microscope was used for the observation.The wild-type and mutant LINC00882 containing miR-214-3p binding sites on the LINC00882 promoter region were ligated into the pMIR luciferase reporter vectors. Then, using Lipofectamine 2000 , we co-transfected the vectors into cells with LINC00882/NC mimic. To normalize the reporter luciferase activities, the dual luciferase reporter assays were carried out. In addition, with mutant-type and wild ATF2 binding sites, the LINC00882 promoter were inserted to pGL3 vector  for promoter assays.9 amount of HepG2 cells were fixed by 4% formaldehyde (v/v diluted by PBS) and the final concentration formaldehyde was 1%. Then, the mM dose of glycine solution was added and cells were cracked by the lysis-buffer. The nuclear sub-fraction of the cells was separated by centrifugation. Then, the nuclear sub-fraction lysates were sonicated to generate an average DNA fragments with the size of 0.5–1 kb. At last, the immunoprecipitation was performed with anti-Tubulin  . The non-specific IgG was used as blank control. The DNA fragments in the complex (Tubulin with Centromere related sequence) was analyzed by the quantitative polymerase chain reaction. The DNA faction amplified was Human (hg38) chr2 q11.1: 92872758- 92873099. The primers are as followed: Forward sequence: 5’-tctgcaagaggatatttggatagc-3’; Reverse sequence: 5’-tcaccataggcctgaaagcg-3’.The ChIP assays were performed following the methods and protocols descripted by the previous publications , 21. Thevia 10% SDS-PAGE and transferred on nitrocellulose membranes . After blocked with 3% BSA , the membranes were incubated with primary antibodies against CENPM , and GAPDH  overnight at 4°C, and secondary antibody labeled with horseradish peroxidase (Abcam) for 2 h at room temperature. Subsequently, the BeyoECL Plus kit  was applied to develop the membranes. GAPDH was used to normalize the expressions of CENPM.Proteins were extracted from HCC cells, and then quantified. Subsequently, 30 μg of proteins was separated in vivo assays, Vital River  provided 4-weeks old male BALB/c nude mice which were randomly divided into two groups: sh-LINC00882-1 group (n = 6) and sh-NC group (n = 6). LINC00882-1 or sh-NC-transfected Huh7 cells were subcutaneously injected into the right flank of nude mice. Digital calipers were applied to measure tumor volume every 4 days, and the formula (tumor volume = 1/2 (length × width2) was used to calculate volumes. 28 days later, the mice were sacrificed, followed by the examination of the weight of all tumors.The experimental procedures were approved by Animal Ethics Committee of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital. For p<0.05 was considered statistically significant.SPSS 19.0 software  was used to analyze all data which are presented as means ± SD. Student’s t-test or one-way analysis of variance was applied to compare the differences between two groups or among multiple groups, followed by Tukey’s tests. A To screen the possible lncRNAs involved in HCC progression, our group searched TCGA datasets and focused on LINC00882 which was distinctly overexpressed in HCC samples (n=371) compared with normal liver samples (n=50) (http://jaspar.genereg.net/), finding that ATF2 may bound to the LINC00882 promoter region with strong probability  to silence the expression of LINC00882. As examined by RT-PCR, the expressions of LINC00882 were significantly inhibited by sh-LINC00882-1 and sh-LINC00882-2 in both HepG2 and Huh7 cells  could disrupt the effect of ATF2, LNC000882 knockdown or miR-214-3p overexpression. Moreover, the overexpression of LNC000882 also repressed the expression of miR-214-3p and enhanced the expression of CENPM. These results further confirmed the specificity of ATF2/LNC000882/miR-214-3p pathway modulate the binding of Tubulin with Centromere related DNA sequence by targeting CENPM.The chromatin immunoprecipitation (ChIP) was performed to detect the binding effect of Tubulin and Centromere related DNA sequence reflecting the influence of CENPM on the centromere-microtubule interaction to form the spindle. As shown in via PDK1/AKT/Caspase 3 pathway , encoding a kinetic protein, modulate chromosomal separation during cellular divisions . The levFor the activity of CENPM itself, we have conducted various studies. Using siRNA to down-regulate the expression of CENPM can significantly inhibit the proliferation of HCC cells. At the same time, for the specific mechanism of CENPM itself, CENPM is a centromere protein, and its expression level is closely related to the normal function of the centromere-spindle. Because cellular immunofluorescence is difficult to quantitatively reflect the direct interaction between tubulin and centromere, we use chromatin immunoprecipitation technology to detect the combination of tubulin and centromere DNA sequence, and finally perform specific effects on CENPM Sexual analysis. Our results show that down-regulating the expression of CENPM can inhibit the binding of tubulin α and β to the centromere sequence. At the same time, the specificity of the action of ATF2/LNC000882/miR-214-3p is determined by setting a reasonable control. Moreover, Tubulin is mainly composed of α- and β-subtypes, while γ-subtypes are mainly distributed in the centrosome. The results of this study also confirmed that in the ChIP experiment, only α-Tubulin and β-Tubulin can interact with centromere DNA sequences, while γ-Tubulin cannot interact with centromere DNA sequences. These methods and results have advantages and innovations: the combination of centromere and tubulin is the basis for the formation of spindles and cell proliferation. The use of chromatin immunoprecipitation technology to detect the combination of tubulin and centromere can be quantitative and intuitive Reflects the interaction between microtubules and centromeres, which is an ideal method for this type of research.However, this study has several limitations. Firstly, the sample size was relatively small, we will collect more samples for research in the future. Secondly, additional mechanisms of LINC00882 such as methylation in regulating HCC progression require further study.Based on results, LINC00882 may be a tumor promotor for HCC, and ATF2 could activate its transcription. LINC00882 exerted oncogenic roles in modulating the proliferation and metastasis of HCC cells. Our findings firstly suggested the underlying mechanisms behind this development, that LINC00882 drove HCC progression by increasing CENPM levels through sponging miR-214-3p, providing a novel therapeutic target for HCC treatments.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.HR, Z-cW, and XC conceived and designed the experiment. HR, Z-cW and Y-xS performed the experiments and wrote the manuscript. C-yQ and W-jZ performed the statistical analyses and generated the figures. HR, WZ, and TL collected the public data. Z-cW and XC collected the patient samples. HR and XC revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Sanming Project of Medicine in Shenzhen(No.SZSM201612063 & No.SZSM201911008) and the Shenzhen Basic Research Program(KJYY20170412153658082).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Transcriptome analysis showed that the upregulated VviDXSs, VviPSYs, and VviCCDs expressions might contribute to the accumulations of terpenes or norisoprenoids in the winter berries of these varieties. Our results provided insights into how climate parameters affected grape volatiles under the double cropping system, which might improve the understanding of the grape berries in response to the climate changes accompanied by extreme weather conditions in the future.The double cropping system has been widely applied in many subtropical viticultural regions. In the 2-year study of 2014–2015, four grape varieties were selected to analyze their fruit volatile compounds in four consecutive seasons in the Guangxi region of South China, which had a typical subtropical humid monsoon climate. Results showed that berries of winter seasons had higher concentrations of terpenes, norisoprenoids, and C6/C9 compounds in “Riesling,” “Victoria,” and “Muscat Hamburg” grapes in both of the two vintages. However, in the “Cabernet Sauvignon” grapes, only the berries of the 2014 winter season had higher terpene concentrations, but lower norisoprenoid concentrations than those of the corresponding summer season. The Pearson correlation analysis showed the high temperature was the main climate factor that affected volatile compounds between the summer and winter seasons. Hexanal, γ-terpinene, terpinen-4-ol,  The grape double cropping system has been applied widely in many subtropical regions . The traIn the double cropping system, bud break was usually enforced between late January and mid-February in the northern hemisphere, resulting in the first bloom in early April and the first crop in June or July. Vines were then pruned and forced again around mid-August, resulting in the second bloom of the side shoots in mid-September and the second crop in mid-January of the following year . Even inFor grape secondary metabolites, the phenolic compositions were the focus of many researchers in dissecting the variations between the summer and the winter berries in the double cropping system . Similar13-norisoprenoids that contribute to fruity, violet, and petrol aromas to grapes and wines (D-erythritol-4-phosphate phosphate (MEP) pathway, carotenoid metabolism, and oxylipin pathway were the most investigated pathways, which could synthesize terpenes, norisoprenoids, and C6/C9 compounds, respectively  and Riesling (R) grapes under the double cropping system . In the The 2-year (2014–2015) study was performed at Guangxi Academy of Agricultural Sciences located in South China . The climate belonged to a subtropical humid monsoon climate with abundant sunshine and heat resources. Vines were trained to a Y-shaped training system with 2 × 4/5 shoots per meter and 1.0 m cordon above ground. Rain shelters were applied to all vines to prevent over-rainfall damage. Four varieties were investigated in this study: CS, R, MH, and V. CS and R grapevines were in the same vineyard, whereas MH and V grapes were in another. The distance between the two vineyards was within 1 km. The detailed information on the variety and phenological stages are shown in The double cropping system in the experiment site was described by Berries of all varieties were sampled four times in each growing season. Sampling time points were as follows: (1) pea-size (E-L 31), (2) onset of veraison (E-L 35), (3) veraison complement (E-L 36), and (4) harvest (E-L 38). There were three biological replicates for each variety. For each replicate, 300 berries were randomly sampled from about 50 vines, which were distributed in three adjacent rows. One hundred berries of which were used in the determination of the physicochemical parameters. The remaining berries were immediately frozen in liquid nitrogen and stored at −80°C for the subsequent metabolite and transcriptome analysis.Meteorological data was acquired from the local climate monitoring station within 1 km away from the experiment site. Photosynthetically active radiation and temperature were recorded per hour. Accumulated rainfall was recorded per day. Growing degree days (base 10°C) was calculated from bloom to harvest according to g) to get clear juice. The total soluble solids (TSS) of the juice were determined with a digital pocket handheld refractometer . The juice pH was determined by a pH meter . TA was measured and expressed as tartaric acid equivalents (g/L) according to the National Standard of People’s Republic of China (GB/T15038-2006).For each replicate, 100 berries were weighted and then manually squeezed. The must was centrifuged . For bound volatile compounds, 2 ml of the clear grape juice sample was added to Cleanert® PEP-SPE resins , which had been activated with 10 ml of methanol and 10 ml of water. Then, the resins were washed with 2 ml of water and 5 ml of dichloromethane to remove water-soluble compounds and free volatiles, respectively. The resins were eluted with 20 ml methanol afterward. The methanol extract was concentrated to dryness by a rotary evaporator under vacuum at 30°C and was redissolved in 10 ml of citrate/phosphate buffer solution . The enzymatic hydrolysis of glycosidic precursors was conducted at 40°C for 16 h by adding 100 μl AR 2000 . The 5 ml of the above sample was added in a 20-ml vial with 1 g NaCl and 10 μl 4-methyl-2-pentanol . Both free and bound samples were placed in a CTC-Combi PAL autosampler  equipped with a 2-cm DVB/CAR/PDMS 50/30 μm SPME fiber  and agitated at 500 rpm for 30 min at 40°C. The SPME fiber was then inserted into the headspace to absorb aroma compounds at 40°C for 30 min and was instantly desorbed into the gas chromatography (GC) injector for 8 min to thermally desorb aroma compounds, and the injection temperature was set at 250°C.The extraction of grapes volatile compounds was according to Both free-volatile and bound-form aroma compounds were extracted by headspace solid-phase microextraction (HS-SPME). Agilent 6890 GC coupled with Agilent 5973C mass spectrometer (MS) was used for the aroma determination. GC was equipped with an HP-INNOWAX capillary column  to separate volatile compounds. The carrier gas was high purity helium with a flow rate of 1 ml/min. The oven program was set as follows: 50°C for 1 min, increased to 220°C at a rate of 3°C/min, and held at 220°C for 5 min. Identification and quantification of volatile compounds followed our research group method . ConcentGSE103226 (CS and R grapes) and GSE168785 (V and MH grapes). Total reads and total mapped reads per sample are shown in The berries of three development stages  in 2014 were selected to determine the transcriptome sequencing. For each replicate, de-seeded 50 berries were ground into powder under liquid nitrogen protection. The following procedures had been described by p < 0.05 (Duncan’s multiple range test or t-test). The Pearson correlation analysis was performed in MataboAnalyst 4.01. The figures were prepared by using GraphPad Prism 8.0.2 , SIMCA 14.1 , and R-3.6.1. Heatmap was prepared by using the “pheatmap” package in R. Principal component analysis (PCA) was performed in SIMCA 14.1 .The SPSS version 22.0  was used for all significance analysis at Meteorological data in the year 2014 and 2015 were shown and discussed by The physicochemical parameters of CS and R grapes were shown and discussed by Totally, there were 173 free-form and 137 bound-form volatile compounds identified in the four grape varieties, and these compounds were sorted into seven groups: C6/C9 compounds, terpenes, norisoprenoids, alcohols, carbonyl compounds, esters, and others , 5. The In CS grapes , two priIn R grapes , two priIn V grapes , two priIn MH grapes , two priTo figure out how the grape-derived aromas changed during the development stages, the total concentrations of C6/C9 compounds, terpenes, and norisoprenoids were calculated, as shown in The C6/C9 compounds were the most abundant aroma compounds in all the grapes. The accumulation trends of C6/C9 compounds were not consistent in the four growing seasons. In most seasons, C6/C9 compounds peaked at E-L 36 and then declined until the harvest, which was in agreement with the previous study . HoweverFor terpenes, MH grapes had the highest concentration among the four varieties, with at least 2000 μg/L at harvest. The other three varieties only had 50–400 μg/L terpenes at harvest, indicating that the grapes of the Muscat family usually had abundant terpenes . CS and For norisoprenoids, there were no consistent results in all varieties. In CS grapes, the summer season berries of 2014 had higher norisoprenoid concentration than those of the winter season in the same year, whereas no significant difference showed in the vintage of 2015. In R grapes, the significant differences between the summer and winter season berries occurred at E-L 35 and E-L 36 in 2014 and at E-L 38 in 2015. MH and V grapes had the same results in the two vintages, and the winter berries had higher norisoprenoid concentrations than those of the summer season.t-test, which showed significant differences in at least one sampling point between seasons. The selected free volatile compounds are shown in To figure out how the growing seasons affected the concentrations of individual volatile compounds, the key compounds were selected by using the E)-β-ionone, 6-methyl-5-hepten-2-one, geranylacetone, (Z)-β-damascenone, and (E)-β-damascenone had lower concentrations in the winter season. β-Damascenone occupied the highest proportion in norisoprenoids of CS grapes (E)-2-hexenal and hexanal were the main C6/C9 compounds with the highest concentrations (E)-2-hexenal than the summer berries at the harvest date.In CS grapes, many terpenes had higher concentrations in the winter season in several or a specific stage, especially in 2014. However, some norisoprenoids, such as (S grapes . (E)-2-htrations . The winD-limonene. Although most norisoprenoids had higher concentrations in several stages, (E)-β-ionone was the only norisoprenoid with a higher concentration in the summer berries in both of the two vintages at harvest. TDN was well-known to contribute “petrol” aromas to “Riesling” wines (E)-2-hexenal and hexanal.In R grapes, most terpenes had higher concentrations in the winter season, such as γ-terpinene, α-terpinene, β-myrcene, terpinolene, geraniol, etc. In 2014, many terpenes only had higher concentrations before harvest in the winter season berries, but these differences disappeared at harvests, such as bornylene, α-terpineol, terpinen-4-ol, α-calacorene, and g” wines , and it D-limonene, β-ocimene, terpinolene, terpinen-4-ol, linalool, α-terpineol, cis-furan linalool oxide, etc. For norisoprenoids, the winter season berries had a higher (E)-β-ionone concentration in all sampling times of 2014 and 2015. (Z)-β-damascenone and (E)-β-damascenone only showed higher concentrations in the winter season berries at harvest in 2014 and 2015. Similar to R grape, its (E)-2-hexenal and hexanal also had higher concentrations in the winter season berries at harvest.In V grapes, most terpenes were also more abundant in the winter season berries, such as In MH grapes, almost all of the selected compounds had higher concentrations in the winter season berries at E-L 38. Many of them did not show any difference in the early development, even were higher in the summer season berries at E-L 35 and E-L 36.Except for terpenes, norisoprenoids, and C6/C9 compounds, some benzene derivatives were also had higher concentrations in the winter season berries in all varieties, such as benzaldehyde, benzeneacetaldehyde, benzyl alcohol, and β-phenylethyl alcohol. These compounds contribute roasted, honey, almond, fruity, and floral flavors to the grapes or their wines . High tecis-furan linalool oxide, and trans-furan linalool oxide showed higher concentrations in the winter season berries in serval stages over 2 years. In R and V grapes, most of the terpenes had higher concentrations in the winter season berries, which was in agreement with the free volatile results. However, in MH grapes, most of the selected compounds showed the opposite trends in 2014 and 2015.The selected bound volatile compounds are shown in r2| > 0.6) in at least three varieties were selected, as shown in cis-furan linalool oxide (G), and trans-pyran linalool oxide (G) were all negatively correlated with high temperatures in all of the four varieties. Other terpenes, such as geraniol, were negatively correlated with high temperatures in R, MH, and V grapes, and geranial (G), nerol (G), geraniol (G), and p-cymene were negatively correlated with high temperatures in R and MH grapes. Different from the pronounced effects of high-temperature hours on aromas, fewer compounds highly correlated with PAR and rainfall were selected. Free- and bound-form p-menthan-8-ol concentrations were all positively correlated with accumulated whole season PAR. trans-Furan linalool oxide (G) was negatively correlated with accumulated whole season PAR in CS, R, and MH grapes. The rainfall correlated compounds had no consistent trends in four varieties.As mentioned above, the summer seasons had more high-temperature hours than the winter seasons, but the accumulated PAR and rainfall were not consistent in the two vintages. Thus, the Pearson correlation analysis was used to figure out how climate factors affect the berries’ volatile compounds at harvest. The highly correlated compounds (| 2 fold change was used to present the variations between the summer and winter season berries through MVA and MEP pathways (Terpenes were derived from two common precursors: isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which were synthesized from two independent pathways: the plastidial MEP and the cytoplasmic MVA pathways, respectively . Carotenpathways .VviDXS genes  were expressed differently in the berries of the winter and summer seasons in at least two varieties. Only the expression of VviDXS2 (VIT_200s0218g00110) was downregulated in MH grapes, and other genes were all upregulated in several varieties in the winter season berries. VviDXS3 (VIT_204s0008g04970) was the common upregulated expression gene in the winter season berries of all four varieties. VviIPI (VIT_204s0023g00600 and VIT_211s0206g00020) was responsible for the transformation between IPP and DMAPP, which had upregulated expression in R and V winter season berries. The mRNA levels of the GPPS small subunit might play a key role in regulating the formation of GPPS and thus affecting the monoterpene biosynthesis  had higher expressions in MH grapes than other varieties and had low expression levels in CS and V grapes  was downregulated in the winter season berries of all four varieties. HMGR was the key enzyme of the MVA pathway. There were three VviHMGR genes  differently expressed in the winter and summer season berries. Among them, VIT_204s0044g01740 was upregulated in R, V, and MH grapes. Terpene synthases (TPSs) were the final enzymes of the terpene biosynthetic pathway. TPS-a, TPS-b, and TPS-g were the main VviTPS genes with high expressions (VviTPS genes were downregulated in the winter season berries. Glycosyltransferases (GTs) could converse free terpenes into their corresponding glycoside bound forms, and three genes had been proved to have such character: VviGT7, VviGT14, and VviGT15 , which expressed differently between the berries of the summer and the winter season. VviPSY3 (VIT_206s0004g00820) and VviPSY2 (VIT_212s0028g00960) had higher expressions in the winter season berries and showed significant differences in several stages. Carotenoid cleavage dioxygenases (CCDs) were the key enzymes that catalyzed the generation of norisoprenoids (apocarotenoids) by cleaving the conjugate double bond of carotenoids  had higher expressions at E-L 35 and E-L 38, whereas it was downregulated at the E-L 36 stage.As mentioned above, the MEP pathway also synthesized carotenoids, which were the precursors of norisoprenoids. The following pathway after synthesizing geranylgeranyl diphosphate (GGPP) was shown in otenoids . There wVviLOXA (VIT_206s0004g01510) had high expression levels in the whole development stages of all the four varieties, which might play a key role in the LOX family  had high expression levels in the development stages. It was reported that VviHPL1 was also related to the accumulation of C6 compounds -2-hexen-1-ol and (Z)-3-hexen-1-ol concentrations than the summer season berries in 2014 and 2015. The winter season berries of CS and R had higher (E)-2-hexen-1-ol concentrations than the summer season berries in 2015 and showed an opposite trend in 2014. The V winter season berries had a lower (E)-2-hexen-1-ol concentration than the summer season berries in both of the two vintages.The C6/C9 compounds, or GLVs, were short-chain alcohols, aldehydes, and esters formed through the oxylipin pathway . The maiX family . The expompounds . Howeverr2| > 0.6) in at least two varieties were selected, as shown in VviDXS3 (VIT_211s0052g01730), which was upregulated in the winter berries in all varieties (VviCCDs (VIT_213s0064g00810 and VIT_213s0064g00840) were positively correlated with the norisoprenoid concentration in R and V grapes, which was in agreement with the previous analysis. However, the two VviCCDs (VIT_213s0064g00810 and VIT_213s0064g00840) were upregulated in the winter berries in R and V grapes.To figure out the relationship between volatile compounds and transcriptome gene expression, we selected the transcriptome genes involved in C6/C9, terpenes, and norisoprenoids synthesis pathway to calculate their correlation with the concentration of each corresponding category during berry development . The highly correlated compounds , and trans-pyran linalool oxide (G) were all negatively correlated with high temperatures in all of the four varieties in the present study. The elevated temperature (>35°C) would inhibit the accumulation of terpenes  suggested that the volatile terpenoid biosynthesis might be decreased by high temperature. Similarly, -D-xylulose-5-phosphate synthase transcripts (VIT_11s0052g01730 and VIT_11s0052g01780), which were required for the isopentenyl pyrophosphate (IPP) synthesis, the universal precursor for the biosynthesis of terpenes. The regression of high temperatures on the VviDXSs expression might be the reason for the lower terpene concentration in the summer season berries in the present study. There were also some gene expressions, such as VviGTs, that were downregulated in the winter season berries. The contents of many bound terpene substances are lower in winter than in the summer season  in CS grapes and other varieties might be the reason.The MH and V grapes had similar results in the two vintages, and the winter season berries had a higher norisoprenoid concentration than those of the summer season. Similar to the terpenes accumulation, high temperatures also inhibited the biosynthesis of norisoprenoids . LecouriVviLOXA was upregulated in the winter berries of R, V, and MH grapes, whereas it was downregulated in CS winter berries. R, V, and MH winter berries had higher C6/C9 concentrations than the summer berries, whereas there was no significant difference in CS grapes at harvest. The variations of VviLOXA expression in CS and other varieties might be the reason.The accumulation trends of C6/C9 compounds were not consistent in the four growing seasons in the present study. In most seasons, C6/C9 compounds peaked at E-L 36 and then declined until the harvest. However, there were some seasons when the berries at E-L 38 had the highest C6/C9 concentrations, such as the winter seasons of V and MH grapes. VviDXSs, VviPSYs, and VviCCDs at the transcription level. The different climates in the summer and winter seasons provided us with a better understanding of how climate changes influenced the grapes’ secondary metabolites. The season variation within a vintage under the double cropping system usually exceeded the effects of the vintage in the traditional viticulture regions, making our results more apparent.This research used metabolomics and transcriptomics to reveal the aroma variations in different grape varieties under the double cropping system. The winter berries had higher TSS content and TA than the summer berries. The lower berry weight in the winter season caused a decreased yield compared to those of the summer season. The winter berries had higher concentrations in many aroma categories than the summer berries, especially terpenes. Climate factor variations were the main reason for the quality variations in the summer and winter season berries. Among all of the climate factors, the temperature might be the dominant one, and its influence could cover up the effects of other factors. Different from other varieties, the CS winter berries had lower norisoprenoid concentrations than the summer berries, indicating that the responses to climate changes might be variety-dependent. The higher concentrations of terpenes and norisoprenoids in the winter berries of most varieties could be associated with the regulated expressions of GSE103226 and GSE168785.The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession numbers FH, JW, and C-QD designed the experiments. W-KC and H-CL performed the experiments. H-CL analyzed the data and prepared the original draft. YW provided the statistical support. X-JB and GC provided the double cropping system vineyards. FH revised the manuscript. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


An acid-fast bacilli (AFB) sputum culture was positive for Nocardia farcinica. Trimethoprim-sulfamethoxazole was started for a Nocardia farcinica pulmonary infection, in addition to antiretroviral therapy. The patient was strongly encouraged to follow-up at the outpatient department.Nocardiosis is an opportunistic infection that most commonly affects immunocompromised patients, with clinical presentations ranging from localized to disseminated disease. In the United States, the reported incidence is approximately 500 to 1,000 cases per year, with an observed male-to-female predominance of 3:1. We present the case of a 37-year-old male with a past medical history of malaria who presented with watery, non-bloody diarrhea for four days associated with a fever for two weeks. The human immunodeficiency virus (HIV) was positive in the emergency room before admission. Computerized tomography (CT) of the chest with contrast revealed an irregular pleural base mass in the right lower lobe with several small air foci. CT of the abdomen and pelvis revealed the right pleural base lung mass to be adherent to the diaphragm, as well as mild splenomegaly. The cluster of differentiation 4 (CD4) count was 9 cells/mm Nocardia species are Gram-positive aerobic rods usually found worldwide in decaying matter, soil, and water. Of the more than eighty identified species of Nocardia, about 25 can potentially cause infection in humans. The Nocardia asteroides complex causes more than 50% of the infections in humans and includes the Nocardia farcinica species [Nocardia infections occur when an immunocompromised host inhales dust particles containing the bacteria, leading to the most common presentation of the disease, namely, pulmonary nocardiosis [ species -2. In th species . Most Noardiosis , 5. We r2) was 98% in room air. The results of the abdominal and rectal exams were unremarkable. The chest x-ray revealed a right lower lung opacity. Computerized tomography (CT) of the chest with contrast demonstrated an irregular, pleural-based mass in the right lower lobe measuring 3.5 x 2.5 cm with several small air foci, likely inflammatory versus infectious in nature , mild lymphopenia (11.5%), microcytic anemia (a hemoglobin of 11 g/dL with a mean corpuscular volume of 79.1 fL), and an elevated high-sensitivity C-reactive protein (CRP) (82.4 mg/L). The patient was started on azithromycin, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. Highly active antiretroviral therapy (HAART) was also started. The acid-fast bacilli (AFB) sputum culture was negative for tuberculosis but returned positive for Nocardia farcinica. Trimethoprim-sulfamethoxazole is the leading first-line conventional therapeutic option for Nocardia farcinica pulmonary infection and no additional antibiotic was started for this patient. The patient was scheduled to follow-up at the Virology Clinic for the HAART and further management. A 37-year-old Guyanese male who immigrated to the United States three years ago, with a medical history of malaria, presented to the emergency department (ED) with complaints of watery, non-bloody diarrhea for four days that began abruptly and occurred about three to four times a day, in association with a subjective fever for two weeks. The result of the Abbott human immunodeficiency virus (HIV) Ag/Ab screening test  done in the ED was found to be positive. The patient denied abdominal pain, chills, night sweats, weight loss, recent travel, sick contacts, recent antibiotic use, nausea, vomiting, or changes in diet. The initial vital signs were as follows: temperature 98.3 ͦ F (36.8 ͦ C), blood pressure 116/69 mmHg, pulse 92 beats per minute (bpm), respiratory rate 8/min, and oxygen saturation . However, both Nocardia farcinica and Nocardia otitidiscaviarum can sometimes be resistant to this treatment. Linezolid, amikacin, imipenem, and fluoroquinolones are the options available for N.farcinica when cultures demonstrate resistance to TMP-SMX [Our newly diagnosed HIV patient with a CD4 count of 9 cells/mmsed host . Althougsed host . It coulsed host . Pulmonaity rate . Earlierity rate . This caity rate . In susp TMP-SMX . In the  TMP-SMX .Nocardia in immunocompromised patients in whom it is unknown. Nocardia could be the initial presentation of an HIV infection. It should be noted that a good medical history, physical examination, and appropriate laboratory testing are essential to rule out other infections and malignancies. A multidisciplinary team is required for dealing with complicated or unclear etiological cases.A high clinical suspicion is required for the diagnosis of


While the sensitivity of the RAT was poor compared with the RT-PCR, the specificity was excellent. However, the sensitivity increased with lower Ct value, and with the right anamnesis the RAT can be a quick and easy approach to distinguish people who are infectious with SARS-CoV-2 from noninfectious people, enabling appropriate triage in clinical practice while waiting for the RT-PCR result.We assessed the performance of a rapid antigen test (RAT) in everyday clinical practice. Between 1 November 2020 until 1 April 2021 all in-patients at the Helios University Hospital Wuppertal, Germany, as well as the accompanying relatives at the Children’s Hospital received a SARS-CoV-2 RAT and a SARS-CoV-2 RT-PCR prior to admission. Out of 3686 patients, 22 (0.6%) subjects were tested positive by RT-PCR and RAT, and 3591 (97.4%) were negative by both methods, showing discordant results: RT-PCR+/RAT− in 58 (1.6%) and RT-PCR−/RAT+ in 15 patients (0.4%). Overall sensitivity and specificity of RAT was 27.5% (95%CI 18.1–38.6%) and 99.6% (95%CI 99.3–99.8%), respectively. The sensitivity was slightly higher in adults  than in pediatric subjects . False negative RAT had a statistically higher Ct-value ( Emerging infectious diseases that spread as pandemics do not only influence the morbidity and mortality of affected populations but also have a major socioeconomic and political impact ,2. SinceSARS-CoV-2 is a single-stranded RNA virus with a virion diameter of 60–140 nm . On the Therefore, a rapid and reliable diagnostic method is crucial in combating the epidemic, as it enables infected individuals to be identified quickly and correctly. This allows for timely isolation and adequate therapy, which contributes to significant containment of the pandemic. It was shown during the SARS-CoV pandemic in 2003 that control measures delayed by one week not only resulted in the outbreak lasting four weeks longer, but that it also increased quantitatively by almost three times .During this pandemic, adults have been the main affected group with higher morbidity and mortality than children ,12,13. FAt our facility, all admitted patients received both reverse transcription polymerase chain reaction (RT-PCR) and a rapid antigen test (RAT) regardless of clinical symptoms or signs of COVID-19. The antigen test can be performed with little effort and provides the result after approx. 15 min, whereas the PCR results take up to 24 h, depending on the capacity of the laboratory ,17.This enables an advanced triage in clinical practice and prevents further infection, as all patients with a positive antigen test are isolated until the (RT-PCR) test result is available.This project was designed to determine the diagnostic test accuracy of the SARS-CoV-2 antigen rapid test in everyday clinical practice and to compare the performance in adult and pediatric subjects.We performed a single center, observational study, which was conducted between 1 November 2020 until 1 April 2021 at the Helios University Hospital Wuppertal, Witten/Herdecke University, Germany. All in-patients, as well as the accompanying relatives at the Children’s Hospital received a SARS-CoV-2 RAT and a SARS-CoV-2 RT-PCR prior to admission. On the day of the test, a form was filled out in advance by the guardians. The form consisted of item parameters regarding clinical symptoms associated with SARS-CoV-2, as well as possible contacts who had been tested positive for the virus. Relatives who had a temperature ≥ 38.5 °C or were under quarantine at the time of admission were not included and had to be replaced by another adult family member.The study was approved by the Ethics Committee of Witten/Herdecke University, Germany (S-09/2021).Each patient admitted to the hospital received a total of two sequential swabs. For each swab, the throat was swabbed first and then the nasopharynx. . One of the swabs was used for the rapid antigen test, while the other was placed in 3 mL of universal transport medium , Xebios Diagnostics GmbH, Düsseldorf, Germany) and sent to our laboratory to carry out the RT-PCR testing.For the RAT, we used the VivaDiag™ SARS-CoV-2 Ag Rapid Test Device (VivaCheck Biotech (Hanghzou) Co., Ltd., Hangzhou, China) and the Panbio™ COVID-19 Ag Rapid Test Device . Both tests were qualitative membrane-based immunoassay based on the Colloidal Gold Immunochromography principle and target the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples.For the isolation of RNA, we used Maelstrom (Taiwan Advanced Nanotech Inc. (TANBead), Taoyuan, Taiwan) or NIMBUS . The RT PCR was performed using different Seegene assays following the manufacturer’s instructions , or assays on the NeuMoDx 288 .Demographic (age and sex) and clinical variables of the study population were obtained from electronic medical records retrospectively.p value of 0.05 or below was considered significant for these comparisons. Statistical analysis was performed with SPSS v26.0 .Continuous variables were given as median with interquartile range (IQR), and the categorical variables as proportions. Differences between groups were compared using the Mann-Whitney U-test or Fisher’s exact test. Receiver operating characteristic (ROC) curves and Youden’s index were used to determine the SARS-CoV-2 RT-PCR cycle threshold (Ct) cut-off value best discriminating between RT-PCR+/RAT+ and RAT− subjects. A two-sided To assess the diagnostic test accuracy of the RAT, the SARS-CoV-2 RT-PCR was established as the gold standard. Sensitivity (Se), specificity (Sp), positive (PPV) and negative predictive value (NPV) were calculated with a 95% confidence interval (CI). Cohen-Kappa statistics were used to calculate the agreement between the various test procedures.A total of 3686 patients were included during this period, 876 (23.9%) of whom were children and 2810 (76.2%) were adults. Of these, 61.3% were female. The median age distribution of the children was 6 years (interquartile range (IQR) 1–14 years) and 43 years (IQR 31–71 years) for the adults. The largest group in this population were adults between 18–34 years old. Clinical characteristics were collected solely in case of positive RT-PCR SARS-CoV-2 result .Out of 3686 patients, 22 (0.6%) subjects tested positive and 3591 (97.4%) tested negative through RT-PCR and RAT. Interestingly, both methods showed discordant results: RT-PCR+/RAT− in 58 patients (1.6%) and RT-PCR−/RAT+ in 15 patients (0.4%)  Table A1The range of cycle threshold (Ct) values of the nucleocapsid (N)-gene were 12.2–39.9 (median 31.9 IQR 24.3–36.8), 14.3–38.5 (median 29.5 IQR 22.6–34.5) for the envelope (E)-gene and 15.9–39.9 (median 30.8 IQR 23.0–36.8) for the RNA-dependent RNA polymerase (RdRP)-gene.p < 0.05) and in the adult population (p < 0.001)  .In our study, ROC curves and Youden’s index analyses indicated that for the N-, E- and RdRP-gene, the optimum cut-off of the different Ct values which discriminated best between RT-PCR+/RAT+ and RT-PCR+/RAT− subjects, were <30, <25 and <30 respectively. The sensitivities were 100% (N- and RdRP-gene) and 88.9% (E-gene). The specificities were 78.6%, 92.9% and 78.6% . In the pediatric group, the Ct cut-off values were <30 (N- and RdRP-gene) and <27 (E-gene) with overall Se and Sp of 100%. For the adult subjects the Ct cut-off values stayed the same with a sensitivity of 100% and specificity of 75% . As expeTo quantify the performance of the RAT in the context of community-based testing, a calculated cut-off for the N-gene Ct value of < 30 was used. This is also the threshold for high virus concentrations that corresponds to the range considered most transmissible. The overall population Se increased to 80% (95%CI 59.3–93.2%) with 75 % (95%CI 50.1–91.3%) for the adult group and 100% (95%CI 47.8–100%) for the pediatric group .The primary goal of controlling the COVID-19 pandemic is to reduce the transmission of infection in the population by reducing the basic reproduction number (R0). The R0 is determined by different factors, such as the duration of infectiousness or the number of susceptible individuals in the population . DiagnosAs of 10 May 2021, the European Commission department of Health and Food Safety has presented a common list of 83 SARS-CoV-2 RAT of which 35 the Member States have agreed to mutually recognize their test results for public health measures [A characteristic of our study population is that accompanying relatives in the children’s hospital who had a temperature ≥ 38.5 °C or had any symptoms related to COVID-19 at the time of admission were excluded. All other subjects from the other medical units were tested, even if they had symptoms of COVID-19 or not. That allowed the calculation of sensitivity and specificity values with greater relevance for daily clinical practice. In our study group, there were more women in the adult group (64.8%), as most of the children were admitted with their mothers.The sensitivity and specificity of the SARS-CoV-RAT were published in a technical data sheet of the test to be 95% and 100% for VivaDiag™ SARS-CoV-2 Ag and 98.1% and 99.8% for Panbio™ COVID-19 Ag, respectively ,23. In tThe overall specificity in this study was very high at 99.6% (99.8% adults vs. 99.1% pediatrics), and similar to that of the manufacturer’s information. This is consistent with previous studies describing the excellent specificities of different RATs ranging from 87.1–100% ,26,27,28However, the sensitivities measured by the manufacturers differed markedly from the value determined by us. Here, the overall sensitivity was 27.5% (30.4% adults vs. 20.8% pediatric). One explanation for this could be that the manufacturer used a large number of samples with a high viral load in the analysis, not reflecting the distribution of the general population.In clinical practice, overall SARS-CoV-2 RAT sensitivity has been reported to vary between 30.2% and 97% depending on the kit used ,28,29,30Our results showed a greater sensitivity in adults (30.4%) than in pediatric subjects (20.8%). While there was no age-related difference in SARS-CoV-2 RNA load in the upper respiratory tract in other studies, one retrospective study in the Netherlands showed that viral load increases significantly with age ,38,39. HIn this study 15 (0.4%) of the RAT results were false positive. In retrospect, it was found that five of these 15 showed up on the same day and that the manufacturer’s instructions were not carried out correctly. Instead of three drops of the extraction solution, more drops were applied. Another source of error for false positive rapid antigen test is explained by the fact that weak bands were often subjectively assessed as positive in order to avoid incorrectly assessing patients as RAT negative and to prevent further spread of the infection. One study found that optimal performance was achieved with significant reductions in false positive rapid antigen tests when the bands were rated positive when spanning the entire width of the strip, regardless of the intensity of the band . A trainThe overall NPV of the SARS-CoV-2 RAT in this study was excellent, at 98.4%, and there was no significant difference in the pediatric or adult subjects, and in cases with low or high Ct-values. Similar high NPVs were also described in previous studies ,41,42. HThe Ct-dependent evaluation of all three genes shows that false negative rapid antigen test had a statically higher Ct value . In thisThe overall increasing of sensitivity to 80% (adults 75% vs. pediatric 100%) for positive SARS-CoV-2 RAT with a Ct value < 30, and thus with higher viral load, has been described in previous studies, with reports going up to 98% ,32,44,456 copies/mL [6 copies/mL were 21.6, 24.5 and 25.2, respectively [However, the cultivation of SARS-CoV-2 does not seem to be possible at a threshold of 10opies/mL . Dependiopies/mL . A quantopies/mL . Using tectively .Nevertheless, in our study, 3% of the potentially infectious people  received negative RAT results. This means that a small number of infectious people were not recognized. In a cohort in Norway with over 4500 subjects tested, this number was 15% [To put this in the context of child public health, serial testing is critical to compensate for the lower sensitivity of RAT, as asymptomatic children with high Ct levels at an early stage of infection may not be detected until later in the course.The strengths of this study lie in the relatively large number of participants, and that it demonstrates the accuracy of a RAT in the context of everyday clinical practice.Nevertheless, the results of the study should be considered in light of its limitations. First, we excluded all relatives who had symptoms associated with SARS-CoV-2 infection, which also explains the low prevalence in our study. The incorrect compliance with instructions and misinterpretation of test results can be interpreted as possible limitations of our study. In addition, we used Ct values as a surrogate for viral load and didn’t determine whether asymptomatic patients with high Ct values were in the early or late stage of infection.To avoid underestimating the viral load of the patient, only efficient sample collection methods can deliver meaningful results. For that, adequate swabs must be made available, especially in pediatrics, which are fine enough to adapt to the child’s anatomy.In summary, we conclude that although the overall sensitivity of the RAT is inferior, the specificity is excellent compared with the RT-PCR in adult and pediatric populations. With the right clinical history, the RAT can be a quick and easy approach, while waiting for the RT-PCR result, to distinguish people who are infectious with SARS-CoV-2 from noninfectious people, and thus enable appropriate triage in clinical practice and reduce further spreading of the virus in the clinical environment. Nevertheless, it is important to note that the excellent NPV of RAT, especially at times of low prevalence, is misleading, and that tools are needed with the highest possible sensitivity in the field.


In the last decades, the widespread use of PSA as the standard tool for prostate cancer diagnosis led to a high rate of overdiagnosis and overtreatment. More recently, multiparametric magnetic resonance imaging (mpMRI) became part of the diagnostic pathway, and several next-generation PSA-based tests  have been proposed. The multivariable approach promises to help with a better stratification of PCa patients at initial diagnosis. In this study, we evaluated the performance of the prostate health index (PHI) and mpMRI for the prediction of positive biopsy and of high-grade PCa at radical prostatectomy (RP). Our findings suggested that PHI had a better ability than mpMRI to predict positive biopsy, whereas a comparable performance in the identification of pathological aggressive PCa was pointed out. Notably, PHI and PHI density might represent useful biomarkers to recognize high-grade PCa in patients with low or uncertain PI-RADS scores on mpMRI.p < 0.001) and PHI density  in the ability to predict positive biopsy with a cut-off value of 42.7 as the best threshold. Conversely, comparing the performance in the identification of clinically significant prostate cancer (csPCa) at RP, we found that PHI ≥ 61.68 and PI-RADS score ≥ 4 were able to identify csPCa (Gleason score ≥ 7 (3 + 4)) both alone and added to a base model including age, PSA, fPSA-to-tPSA ratio and prostate volume. In conclusion, PHI had a better ability than PI-RADS score to predict positive biopsy, whereas it had a comparable performance in the identification of pathological csPCa.Widespread use of PSA as the standard tool for prostate cancer (PCa) diagnosis led to a high rate of overdiagnosis and overtreatment. In this study, we evaluated the performance of the prostate health index (PHI) and multiparametric magnetic resonance imaging (mpMRI) for the prediction of positive biopsy and of high-grade PCa at radical prostatectomy (RP). To this end, we prospectively enrolled 196 biopsy-naïve patients who underwent mpMRI. A subgroup of 116 subjects with biopsy-proven PCa underwent surgery. We found that PHI significantly outperformed both PI-RADS score (difference in AUC: 0.14;  Multiparametric magnetic resonance imaging (mpMRI) of the prostate currently plays a central role in the diagnostic pathway of suspected prostate cancer (PCa)  and is aWe performed a monocentric, prospective and observational study between May 2020 and July 2021 to assess whether PHI can improve the identification of csPCa in patients who underwent mpMRI. Patients underwent a standard prostatic biopsy, which consisted of at least 16 needle biopsy cores obtained under transrectal ultrasound (TRUS) guidance.Patients with PI-RADS ≥ 3 received a targeted biopsy with 12-core standard systematic biopsy. PSA values included between 2 and 10 ng/mL and/or a suspicious digital rectal exploration without a previous prostate biopsy. Subjects suffering from acute prostatitis or urinary tract infections, subjects with previous surgery or biopsy and subjects using 5-α reductase inhibitors were excluded. Prostate volume was calculated by transrectal ultrasound (TRUS).Primary and secondary Gleason scores were assigned by a single genitourinary pathologist blinded to the biomarker values, according to the 2005 consensus conference of the International Society of Urological Pathology definitions .Among the 196 enrolled patients, 116 with biopsy-proven PCa underwent robot-assisted radical prostatectomy (RALP) at the Division of Urology of the University Federico II  within 3 months. None of the study patients received neoadjuvant hormonal therapy  and/or other drugs  that could alter the PSA values. For the definition of csPCa, we used postoperative Gleason score ≥ 7 (4 + 3) ,28. InfoParticipants had blood drawn before DRE at each visit. Whole blood was allowed to clot before serum was separated by centrifugation. Serum aliquots were stored at −80 °C until samples were processed, according to Semjonow et al. . SpecimempMRI was performed using a 3-T MRI scanner Siemens MAGNETOM Vida 3 T  acquiring diffusion-weighted imaging (DWI), dynamic contrast enhancement imaging (DCE), T1-weighted axial and T2-weighted triplanar imaging. All mpMRI images were independently interpreted by four different experienced genitourinary radiologists, with at least 5 years of experience, according to PI-RADS version 2.0. The images were segmented to obtain and record lesion locations and PI-RADS scores.t-test for independent samples, the nonparametric Mann–Whitney test, the chi-square test or the Fisher test where appropriate. Discrimination ability of the different PSA-derived biomarkers, as well as PI-RADS score, was assessed using ROC curve and further quantified computing the area under the curve (AUC) with the corresponding 95% confidence interval (95% CI). Optimal thresholds were defined as those maximizing the Youden index (sensitivity + specificity − 1). Multivariable logistic regression models were built to assess the independent and incremental capacity of PHI, PHI density and PI-RADS score in identifying PCa and csPCa with respect to a set of base clinical variables including age, PSA, fPSA-to-tPSA ratio and prostate volume. All statistical comparisons involving two AUCs, as well as the estimation of the 95% CIs of sensitivity and specificity associated with the optimal thresholds, were based on bootstrap resampling. Statistical analyses were performed using the R language . p-values < 0.05 were considered statistically significant.Standard descriptive statistics were used to characterize the overall sample: mean ± standard deviation or median  with range in case of numerical variables and absolute frequencies with percentages in case of categorical factors. Between-group differences were evaluated using The overall cohort consisted of 196 patients with a mean age of 66.6 ± 7.8 years (range: 48 to 85 years). Mean PSA was equal to 6.2 ± 2 ng/mL (range: 2 to 10 ng/mL); PI-RADS score was equal to or greater than 4 in 111 patients (56.6%) and equal to 3 in 44 patients (16.8%). Median PHI was equal to 51.1 (range: 12.7 to 179.3) .p < 0.001) and PHI density  . All PSA-derived biomarkers, as well as PI-RADS score, were significantly associated with the risk of PCa at univariate analysis with an AUC ranging from 0.65 (95% CI: 0.559 to 0.742) for free PSA to 0.969 (95% CI: 0.948 to 0.990) in case of PHI . In the = 0.002) . The opt= 0.002) .Among the 142 patients with positive biopsy, prostatectomy data were available for a subset of 116 patients (81.7%) with a mean age of 65.6 ± 6.9 years (range: 48 to 81) .p = 0.536). The optimal cut-off for the identification of csPCa at prostatectomy remained unchanged for PI-RADS , whereas it became higher for PHI   or higher) was observed in 90 patients (77.6%). Only PHI (as well as the ratio between p2PSA and free PSA) and PI-RADS score, but not PHI density, preserved a significant association with the presence of csPCa at surgery with an AUC equal, respectively, to 0.747 (95% CI: 0.637 to 0.857) and 0.709 (95% CI: 0.593 to 0.825) . However to 100) .p = 0.044 with respect to the base model); by adding PI-RADS to the base variables, an almost equal increase was observed  . By adding PHI to the set of base variables, the AUC of the model was raised to 0.784  .In the overall cohort of 196 patients, both PHI and PHI density showed significant differences among the classes of PI-RADS score. Patients with PI-RADS score ≤ 2 and subjects with PI-RADS equal to 3 presented significantly lower PHI values than patients with a PI-RADS score equal to 4 or 5 . An almoIn addition, PHI values higher than 42.7  and PHI n = 196) centered on the evaluation of PHI and mpMRI findings according to PI-RADS version 2, which included not only patients who underwent biopsy but also a subgroup (n = 116) of subjects treated by RP. Thus, our study population also allowed us to evaluate the ability to recognize the presence of pathological csPCa.Novel biomarkers and MRI are well-known tools useful to identify csPCa ,20,22. Tn = 35) to avoid a targeted biopsy, with both sensitivity and NPV of 100% [n = 249) and repeated (n = 144) biopsy subgroups. The authors also showed that adding PHI to PI-RADS significantly increased the accuracy for the prediction of any cancer and csPCa in both the subgroups. Recently, Kim et al. [We found that PHI significantly outperformed both mpMRI and PHI density in the prediction of positive biopsy. A cut-off value of 42.7 has been identified as the best threshold. In addition, we compared the ability of PHI, PHI density and mpMRI to predict the presence of csPCa at RP. We found that PHI ≥ 61.68 and PI-RADS score ≥ 4 were able to identify csPCa (Gleason score ≥ 7 (3 + 4)) both alone and added to a base model including age, PSA, fPSA-to-tPSA ratio and prostate volume. We also observed that p2PSA/fPSA, but not PHI density, had the same ability as PHI in the recognition of csPCa. Finally, we demonstrated a good correlation of PHI and PHI density with PI-RADS score. Of note, increased PHI and PHI density values allowed identifying csPCa in subjects with PI-RADS score ≤ 3 corresponding to negative or uncertain mpMRI outcomes. Our findings suggested that PHI and mpMRI showed a comparable ability to predict csPCa defined at RP. A sequential or combined use of the test and imaging might ensure the best ability to reduce unnecessary biopsies without missing aggressive PCa as previously demonstrated by other authors. In the first report of the complementary role of PHI and mpMRI in repeated biopsies , the aut of 100% . In a st of 100%  includinm et al.  showed tThe proportion of men who harbor csPCa was very different in PI-RADS 3 and in PI-RADS 4/5 . PI-RADSA low number of csPCa cases remained undiagnosed by negative mpMRI. PI-RADS 1–2 lesions corresponded to about 1 in 10 probability of diagnosis of csPCa . The resDall’Era  suggesteWe recently demonstrated that by using artificial neuronal networks (ANNs), it is possible to develop models combining different PSA-derivatives  optimizing high-grade PCa recognition . Taking To the best of our knowledge, this study was the largest prospective cohort study aiming to evaluate PHI and imaging as partners in crime in the battle against overdiagnosis and overtreatment, which included not only a biopsy-naïve patient setting but also a subgroup that underwent surgery. Thus, one strength of this study is the definition of csPCa based on pathological grading, whereas one limitation was that our results may not be extended to repeated biopsy. Another limitation is that the subgroup of PI-RADS 3 was small, and the results need further studies on a larger number of patients with uncertain mpMRI outcomes, since the role of PHI in this group is a relevant issue to reduce unnecessary biopsies due to mpMRI failure. In addition, a major limitation of this study is that we did not evaluate the results of the follow-up of patients with high PI-RADS score and negative biopsy. This issue is clinically relevant, and thus we are currently collecting the data for a future study.In conclusion, our findings suggested that PHI had a very good accuracy as a noninvasive biomarker to predict positive biopsy. PHI and mpMRI had a comparable performance in the recognition of pathological csPCa. PHI and PHI density were able to improve pathological csPCa identification in patients with negative or uncertain mpMRI outcomes. The implementation of PHI in addition to mpMRI in clinical practice may ensure a personalized therapeutic approach for patients with suspected PCa and may bridge the gap of PI-RADS 3 mpMRI outcomes.